Although members of the NIMA-related kinase (NEK) family have been implicated extensively in human dis- eases including cancer and renal and cardiovascular defects, their physiological substrates are largely un- known. Moreover, little is known about the upstream and downstream components of NEK signaling pathways or about the proteins that modulate NEK activities. This has led to a fundamental gap in our knowledge concerning the molecular and cellular functions of this biomedically relevant and highly conserved family of protein kinases. The long-term goal is to characterize a recently discovered and potentially widespread function for NEKs in intracellular trafficking and extracellular matrix remodeling. The objective of this application isto characterize the trafficking functions of NEKL-2/NEK8 and NEKL-3/NEK6/7 during C. elegans development and in human cell culture systems. The central hypothesis is that NEKs regulate endocytosis and vesicle trafficking through interactions with components of the cytoskeleton including microtubule (MT)-associated proteins, molecular motors, and regulators of the acting network. Strong preliminary data support the two specific aims: 1) Elucidate the mechanisms by which NEKL-2 and NEKL-3 control trafficking; and 2) Identify molecular targets and components of the NEKL signaling pathway. Under Aim 1, the localization of NEKLs to intracellular traf- ficking and cytoskeletal compartments will be characterized, and the specific cellular functions of NEKLs in regulating vesicular trafficking and cytoskeletal organization and dynamics will be determined. In addition, the hypothesis that ankyrin repeat (AR) binding partners of NEKLs act as signaling scaffolds will be tested. Finally, a panel of assays will be carried out to test the rles of NEK6/7, NEK8, and associated AR proteins in trafficking functions in mammalian cells. Under Aim 2, proven RNAi-feeding screens will be carried out to identify components and candidate targets of the NEKL signaling networks. In addition, a recently developed powerful chemical genetic-proteomic strategy, which is well supported by preliminary data, will be used to directly identify in vivo substrates of the NEKLs. Follow-up studies will be carried out on select validated targets to deter- mine their functions in trafficking and cytoskeletal organization downstream of the NEKs. The approach is in- novative because other groups have not applied powerful forward genetic methods to study NEK kinases and because the function of NEKs in intracellular trafficking is entirely unknown. In addition the proposed chemical- genetic strategy to identify substrates has not been previously applied to C. elegans or to the study of NEKs in any system. The proposed research is significant because human NEKs are widely implicated in human dis- eases and have been suggested as drug targets, but their roles during interphase are not well understood nor, in most cases, are their physiological substrates known. As an additional benefit, these studies will lead to the characterization of previously unknown but conserved regulators of intracellular trafficking and extracellular matrix remodeling, both of which are highly relevant to human development, health, and disease.